Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany.
Department of Molecular Microbiology, John Innes Centre, Norwich, UK.
Microbiology (Reading). 2010 Oct;156(Pt 10):3166-3179. doi: 10.1099/mic.0.042572-0. Epub 2010 Jul 1.
Streptomyces coelicolor A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three narGHJI operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple narGHJI mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenum-dependent. Analysis of S. coelicolor growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.
变铅青链霉菌 A3(2)合成三种膜结合的呼吸硝酸盐还原酶 (Nars)。在液体培养基中好氧生长时，细菌能够将 50 mM 硝酸盐化学计量地还原为亚硝酸盐。构建并分析了一个缺失所有三个 narGHJI 操纵子的突变体，表明它不能还原硝酸盐。缺失编码 MoaA 的基因，该基因催化钼辅因子生物合成的第一步，也阻止了硝酸盐的还原，这与 Nars 是钼酶一致。与三重 narGHJI 突变体相反，moaA 突变体也不能将硝酸盐作为唯一氮源，这表明变铅青链霉菌中的同化硝酸盐还原酶也是依赖钼的。在固体培养基上分析变铅青链霉菌的生长表明，Nar 活性存在于孢子和菌丝（菌丝体）中。用硝酸盐类似物氯酸盐开发的生存测定表明，野生型变铅青链霉菌孢子和菌丝在厌氧孵育后对氯酸盐敏感，与硝酸盐的存在无关，而 moaA 和三重 nar 突变体都对氯酸盐具有抗性。用 cosmid 上携带的单个 narGHJI 操纵子对三重 nar 突变体进行互补表明，每个操纵子编码的酶都能在硝酸盐或氯酸盐还原中被合成并具有活性。从这些研究中获得的数据允许将 Nar1 活性暂定分配给孢子，Nar2 分配给孢子和菌丝体，而 Nar3 专门分配给菌丝体。
